scholarly journals Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha

1980 ◽  
Vol 8 (1) ◽  
pp. 99-110 ◽  
Author(s):  
George E. Wright ◽  
Earl F. Baril ◽  
Neal C. Brown
Keyword(s):  
Hela Cell ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Selective Inhibitor ◽  
Dna Polymerase Alpha

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

scholarly journals Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases

1979 ◽  
Vol 178 (3) ◽  
pp. 621-626 ◽  
Author(s):  
J F Burke ◽  
P M Duff ◽  
C K Pearson
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Acid Synthesis ◽  
Cell Nuclei ◽  
Isolated Nuclei ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta ◽  
Deoxyribonucleic Acid Synthesis

In order to ascertain the identity of the DNA-dependent DNA polymerase responsible for the observed DNA synthesis in nuclei isolated from baby-hamster kidney (BHK-21/C13) cells a comparative study was carried out on the effects of some drugs, reported to influence DNA synthesis, on DNA synthesis catalysed by these nuclei and by partially purified DNA polymerase-alpha and -beta. In all cases DNA synthesis by isolated nuclei and polymerase-alpha was inhibited to similar extents by N-ethylmaleimide, p-hydroxymercuribenzoate, novobiocin, heparin and phosphonoacetic acid; polymerase-beta was much less affected by these compounds. Ethidium bromide inhibited all DNA synthesis to similar extents, although at low concentrations (about 2 microgram/ml) synthesis in isolated nuclei was stimulated. The results are discussed in relation to the proposal that DNA polymerase-alpha catalyses the covalent extension of Okazaki fragments that these nuclei carry out in vitro.

scholarly journals Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 256-264 ◽  
Author(s):  
V Gandhi ◽  
E Estey ◽  
MJ Keating ◽  
A Chucrallah ◽  
W Plunkett
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribonucleotide Reductase ◽  
Acute Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Biochemical Modulation ◽  
Dna Polymerase Alpha ◽  
Human Dna ◽  
Acute Myelogenous ◽  
Dna Primers

Abstract The effectiveness of arabinosylcytosine (ara-C) for the treatment of acute myelogenous leukemia (AML) depends on the formation of its active metabolite, the triphosphate of ara-C (ara-CTP). Using biochemical modulation strategies to increase the accumulation of ara-CTP in leukemia blasts, a clinical protocol was designed combining 2- chlorodeoxyadenosine (CdA), an inhibitor of ribonucleotide reductase, and ara-C for adults with AML. The protocol stipulated an infusion of 1 g/m2 of ara-C over 2 hours on day 1. A continuous infusion of CdA (12 mg/m2/d) begun 24 hours later and continued for 5 days. Identical doses of ara-C were administered on days 3, 4, 5, and 6. Pharmacokinetic and pharmacodynamic interactions between CdA and ara-C during therapy were investigated. To complement these studies, molecular actions of the triphosphate of ara-C and CdA on DNA extension by human DNA polymerase alpha in an in vitro model system was conducted. In the circulating leukemia blasts of 7 of the 9 patients studied, ara-CTP pharmacokinetics showed a median 40% increase in the rate of ara-CTP accumulation after 24 hours of CdA infusion. The ex vivo effect of CdA on accumulation of ara-CTP in AML blasts was similar to that during therapy except that the enhancement was less. The DNA synthetic capacity of the circulating blasts was inhibited to a greater extent by administration of CdA and ara-C in combination than by either one alone. Additionally the lowered level of DNA synthesis was maintained until the next infusion of ara-C. Endogenous levels of deoxynucleotides increased 24 hours after ara-C infusion. Administration of CdA in general lowered the concentrations of all dNTPs. DNA pol alpha incorporated CdATP and ara-CTP with high affinity in a DNA primer extending over an oligonucleotide template of defined sequence. Human DNA polymerase alpha extended DNA primers terminated by CdA monophosphate (CdAMP) at its 3′-end by incorporating ara-C monophosphate (ara-CMP). The tandem incorporation of CdAMP and ara-CMP resulted in nearly complete inhibition of DNA primer extension. The insertion of two analogs in sequence, inhibition of ribonucleotide reductase, and the metabolic potentiation of ara-CTP by CdA infusion may be responsible for sustained inhibition of DNA synthesis in the circulating leukemia blasts during therapy with this combination regimen.

scholarly journals Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

1991 ◽  
Vol 11 (4) ◽  
pp. 2108-2115 ◽  
Author(s):  
K L Collins ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Dna Polymerase Alpha ◽  
Primase Complex

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

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